We are investigating the role of protein kinase C phosphorylation in the regulation of contractile proteins in both smooth muscle and nonmuscle cells. The sequence of the protein kinase C phosphorylation site in the turkey gizzard myosin light chain has been nearly determined. It appears to be either threonine 9 or 10 at the N-terminal portion of the light chain. In two-dimensional tryptic peptide maps of the phosphorylated heavy meromyosin and light chain, we have found that there are two major peptides that are phosphorylated. This is in contrast to previous work in this laboratory that indicated that there was only one major phosphorylated tryptic peptide in HMM. We have also successfully phosphorylated the nonmuscle myosin isolated from human platelets so as to incorporate 1 mole of phosphate per mole of light chain. The kinetics of phosphorylation appear to be similar to that seen with the smooth muscle myosin from turkey gizzard. In preliminary studies, we have shown that smooth muscle phosphatase I will dephosphorylate HMM, the soluble two-headed subfragment of myosin, that has been phosphorylated with protein kinase C. This phosphatase does not have activity against HMM phosphorylated with myosin light chain kinase.